Major congenital malformations occur in up to 10% of babies born to women with type 1 or 2 diabetes resulting in a significant public health problem. Congenital malformations during maternal hyperglycemia are the result of excess apoptosis in target tissues. Maternal hyperglycemia activates pro-apoptotic cascades resulting in excess apoptosis in embryonic cells leading to embryonic dysmorphogenesis. Our published data in caspase 8 and JNK2, preliminary data in amelioration of diabetic embryopathy by Foxo3a deficiency and preventive effect of phytochemical EGCG on hyperglycemia-induced malformations via inhibition of Foxo3a activation, implicate a Foxo3a centric pro-apoptotic cascade in this disease process. We hypothesize that a JNK1/2, Foxo3a, TRADD, caspase 8 pathway acts to enhance apoptosis, and that Foxo3a is a key activator of TRADD transcription. TRADD then induces apoptosis in the neuroepithelium of the developing embryo leading to neural tube defects (Neural tube defect, NTD) characteristic of the disease (Fig. 1). Phytochemical EGCG reduces diabetes-induced NTD via blockade of this pathway. In Specific Aim 1, we will determine if Foxo3a is a key downstream target of JNK1/2 in the pathway leading to hyperglycemia-induced embryonic malformation. We will dissect the detailed mechanisms whereby diabetes-induced Foxo3a activation in connection with JNK. We will monitor cytoplasmic/nuclear Foxo3a phosphorylation state, Foxo3a and 14-3-3 interaction, nuclear translocation and DNA binding under JNK2 deficiency. We will determine if Foxo3a is required for hyperglycemia-induced apoptosis and embryonic malformation (Aim 2). We hypothesize that Foxo3a activity is required for activation of TRADD expression. We will use Foxo3a knockout (Foxo3aKO) mice to test whether Foxo3a is required for TRADD expression, caspase-dependent apoptosis, and embryo malformation. We will determine if TRADD is required for apoptosis in maternal hyperglycemia-induced embryopathy, and the effect of EGCG on diabetic embryopathy and the diabetes-induced pro-apoptotic pathway (Aim 3). TRADD-FADD complex triggers caspase 8 activation leading to apoptosis. We will use ?-actin-FADD-DN (Dominant Negative) transgenic mice to test whether blockade of TRADD function prevents hyperglycemia-induced malformation, caspase 8 activation and apoptosis. Using non-diabetic and diabetic pregnant mice, we will determine EGCG's effects in vivo by administering dietary EGCG supplements. We will determine EGCG's effects on maternal diabetesinduced NTD, phosphorylation of JNK1/2 and Foxo3a, Foxo3a nuclear translocation, upregulation of TRADD, caspase 8 cleavage and apoptosis. Caspase 8 is one of the apoptosis initiator being identified in diabetic embryopathy and its activation leads to activation of Bcl-2 family members and effector caspases such as caspase 3. We further define the transcription factor and the apoptotic gene mediating diabetes-induced caspase 8 activation and apoptosis using our previous findings in Bcl-2 and caspase 3 as endpoints. Using elegant genetically modified mouse models in such a complex disease would have high impact in this field. To study the effect of EGCG, we bridge our mechanistic studies to a possible therapeutic candidate. The innovations of our studies and approaches include the critical role of Foxo3a among other Foxo factors, potential translational EGCG studies, well-designed use of genetically modified mice and first defining the detailed mechanisms whereby diabetes-induced Foxo3a activation PUBLIC HEALTH RELEVANCE: Major congenital malformations such as neural tube defects occur in up to 10% of babies born to women with type 1 or 2 diabetes resulting in a significant public health problem. The proposed study is to identify apoptotic intermediates responsible for the induction of diabetic embryopathy and define the mechanism of diabetic embryopathy at both the cellular and transcriptional levels. By unraveling the mechanisms leading to diabetic embryopathy, the results of the study will provide a mechanistic basis for the use of cutting-edge, mechanism-based therapeutic strategies designed to prevent diabetes-associated birth defects.